Significance of Pituitary Leptin to Gonadotropes

描述(申请人提供)：脑下垂体瘦素呈周期性变化，表明它可能促进或允许促性腺激素的分泌。在缺乏食物的情况下，垂体瘦素和黄体生成素的下降是平行的，在葡萄糖的调节下，脑垂体的瘦素和黄体生成素也同时恢复。这一间接证据表明，如果营养状况最佳，脑下垂体瘦素可能是允许促性腺激素功能的代谢通道。这项拟议的初步研究旨在建立细胞和动物模型，以检验生长激素分泌的垂体瘦素对促性腺激素释放激素(GnRH)的正常发育和反应至关重要的假设。这些研究将在目标1中用体外方法检验这一假设，在该方法中，瘦素mRNA的siRNA将被用来敲除脑下垂体瘦素。在目标2中，将通过体内研究来验证这一假设，在体内研究中，通过Cre-loxP技术删除生长激素中瘦素的表达。为了去除生长激素中的瘦素基因，携带转基因rGHp-Cre的突变小鼠将与携带瘦素基因两侧的loxP的小鼠杂交。在这两组AIMS中，将测试促性腺激素对GnRH的反应，以确定在体外或体内(在生长激素中)切除垂体瘦素是否会影响促性腺功能。促性腺激素的发育和生育能力也将在生长激素中剔除瘦素的突变小鼠身上进行测试。这项初步研究提供了一种有重点的方法来回答垂体瘦素对促性腺激素功能的重要性的问题，避免了全球瘦素基因敲除所出现的影响促性腺激素的代谢问题和对瘦素功能的混乱解释。这项先导性研究将为未来的研究奠定基础，这些研究将测试脑下垂体瘦素对其他调节剂(类固醇、神经肽Y和激活素)的促性腺激素反应的影响。未来可以利用Cre-loxP技术进行体内研究，以测试其他类型的垂体细胞(促肾上腺皮质激素和促性腺激素)产生的瘦素的重要性。这项研究的最终目的将是确定瘦素如何支持生殖能力，以及有关营养不良的信息可能如何与不孕不育联系起来。 公共卫生相关性：这项拟议的初步研究重点是脑下垂体瘦素对促性腺激素的重要性；检验脑下垂体瘦素对促性腺激素发育和/或功能至关重要的假设。目的1将建立一个体外模型；该研究将使用针对瘦素mRNA的siRNA来去除垂体瘦素，以确定其对促性腺激素功能的影响。目的2利用Cre-loxP技术建立体内转基因小鼠模型，利用Cre-loxP技术在生长激素细胞中特异性地去除瘦素。在生长激素中剔除瘦素的转基因小鼠将接受青春期开始时间、生育能力和促性腺激素对刺激的反应的测试。这些研究的重点是将营养状况与生育力联系起来的基本调控机制。

DESCRIPTION (provided by applicant): Pituitary leptin changes in a cyclic manner that indicates it may facilitate or permit the secretion of gonadotropins. There are parallel reductions in pituitary leptin and LH during food deprivation and a parallel recovery mediated by glucose. This circumstantial evidence suggests that pituitary leptin may serve as a metabolic gateway to permit gonadotrope function, if the nutritional status is optimal. This proposed pilot study is designed to develop cellular and animal models that test the hypothesis that pituitary leptin from somatotropes is vital for the normal development and responses of gonadotropes to gonadotropin releasing hormone (GnRH). The studies will test the hypothesis with in vitro approaches in Aim 1 in which siRNA for leptin mRNA will be used to knock out pituitary leptin. In Aim 2, the hypothesis will be tested by in vivo studies, in which leptin expression is deleted in somatotropes by Cre-loxP technology. To ablate the leptin gene in somatotropes, mutant mice bearing the transgene rGHp-Cre will be crossed with mice that bear loxP flanking the leptin gene. In both sets of aims, gonadotrope responses to GnRH will be tested to determine if ablation of pituitary leptin in vitro or in vivo (in somatotropes) affects gonadotrope functions. Gonadotrope development and fertility will also be tested in the mutant mice that have leptin excised in somatotropes. This pilot study provides a focused approach to answer the question of the significance of pituitary leptin to gonadotrope functions, which avoids metabolic problems seen with global leptin knockouts that affect gonadotropes and confound interpretation of leptin functions. The pilot study will lay the foundation for the development of future studies that test the effect of pituitary leptin on gonadotrope responses to other regulators (steroids, neuropeptide Y and activin). Future in vivo studies with Cre-loxP technology can be proposed to test the significance of leptin produced by other pituitary cell types (corticotropes and gonadotropes). The ultimate aim of the study will be to determine how leptin supports reproductive competence and how information about malnutrition may be linked with infertility. PUBLIC HEALTH RELEVANCE: This proposed pilot study is focused on the significance of pituitary leptin to gonadotropes; testing the hypothesis that pituitary leptin is vital for gonadotrope development and/or functions. Aim 1 will develop an in vitro model; the studies will use siRNA for leptin mRNA to ablate pituitary leptin to determine its effects on gonadotrope function. Aim 2 will develop an in vivo transgenic mouse model in which leptin is ablated specifically in somatotropes by Cre-loxP technology. Transgenic mice, in which leptin is knocked out in somatotropes, will be tested for the time of onset of puberty, fertility, and responses by gonadotropes to stimuli. These studies focus on basic regulatory mechanisms that link nutritional status to fertility.